Functional in vitro diversity of an intrinsically disordered plant protein during freeze–thawing is encoded by its structural plasticity

Abstract Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration‐sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra‐ and inter‐monomeric helix–helix interactions, demonstrate how oligomerization is driven by an α‐helical molecular recognition feature (α‐MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right‐handed coiled‐coils might be a recurring theme for homo‐ and hetero‐oligomerization of LEA proteins.

BSA.The symbols represent the SANS data at low q angles in the form of Guinier plots, with solid lines depicting fits, which were used for calculation of RG.

Fig. S2 :
Fig. S2: Neutron scattering data of COR15A in increasing volume fractions of the proteinaceous crowder

Fig. S3 :
Fig. S3: Neutron scattering data of COR15A in increasing volume fractions of the proteinaceous crowder BSA expressed as normalized Kratky plots.The dashed lines indicate a peak position at qRG = 3 0.5 , which is expected for a folded globular protein.

Fig. S4 :
Fig. S4: Expression controls of plants carrying either the COR15A, 4GtoA or G66A sequence in expression sites 1 and 2 and the empty vector as a negative control, referring to BiFC data in Fig. 2. Both putative interaction partners in the raw protein lysates were immunodetected with anti-HA and anti-Myc antibody, respectively.

Fig. S5 :
Fig. S5: RMSD (A-D), intra-(F-I) and intermolecular (K-N) H-bonds and secondary structure in terms of the ratio of random coil (P-S) and α-helix (U-X) of the monomeric COR15A variants during 120 ns MD simulations.Error bars depict the standard deviation of 10 simulation repeats.(E), (J), (O), (T) and (Y) show the average slopes of the respective parameter during the first ns of MD simulation for all COR15A variants in direct overlay.

Fig. S6 :
Fig. S6: Distance between the residues involved in most contacts as depicted in Fig. 3J is stable throughout simulation time

Fig. S8 :
Fig. S8: X-ray scattering data of COR15A variants without glycerol (black symbols) and at 70 % glycerol (red symbols).The symbols represent the experimental data.The respective data for COR15A have been published previously (1).(A-C) show the SAXS data in log-log representation with solid lines depicting fits using generalized Gauss functions.(D-F) represent the SAXS data at low q angles in the form of Guinier plots, which were used for calculation of RG. (G-I) express the SAXS data as normalized Kratky plots.The dashed lines indicate a peak position at qRG = 3 0.5 , which is expected for a folded globular protein.

Fig. S11 :
Fig. S11: CF leakage from ICMM liposomes after a freeze-thaw cycle to -20 °C.ICMMs were frozen in increasing concentrations of COR15A and its mutants at 12 different liposome surface occupancies.Boxes are plotted as a function of ICMM surface occupancy in order to account for size differences between the COR15A variants and depict median and quartiles.Whiskers represent 10 and 90% percentiles and the symbols 5 and 95% percentiles.Solid lines depict the regression curves from fitting the raw data to a dose response model as shown in Fig.6B.

Fig. S12 :Fig. S13 :
Fig. S12: Light scattering intensities of 0.15 g/L LDH in 30 mM sodium phosphate buffer, pH 7.4 alone or in the presence of the COR15A variants or the control protein RNase A in concentrations referring to LDH surface occupancies of 4-5 after zero to five freeze-thaw cycles in liquid nitrogen.Scattering intensities were measured in a custom built light scattering instrument and normalized to toluene as internal reference.

Fig. S14 :
Fig. S14: Overlay of monomer (left) and dimer (right) structures of COR15A modelled and docked by I-TASSER and Haddock (silver) and AlphaFold (gold).The RMSD between monomers and dimers modelled by I-TASSER and AF was 5.3 A and 6.9 A, respectively.

Table S1 :
List of cloning primers